The vertebrate immune response is largely dependent on a vast population of antigen- receptors. Much of the diversity in these molecules is now understood to arise from a site-specific DNA recombination process called V(D)J recombination. Inherited coding regions are cut and pasted in a reaction that assembles mature genes. The combinatorial and mechanistic contributions to the product create a degree of diversity that could not be obtained by inheriting preformed genes. Two proteins, RAG1 and RAG2, play an essential role in the reaction. In the past two years, these two proteins have been shown to cut the target DNA at the appropriate site in an in vitro reaction that contains no other factors. A detailed study of the role of these two proteins alone and in concert with other proteins in the later steps of the reaction is vital to an understanding of this critical mechanism.
The specific aims are: (1) Determine the contacts between individual RAG proteins and DNA. A DNA binding assay based on UV crosslinking will be used to identify the DNA binding domains of the two proteins. The contact sites will be determined on the DNA and on the protein. Definition of the DNA binding domain will also allow better understanding of the architecture of the two proteins, and delineation of domains that should be involved in protein-protein contacts. (2) Demonstrate protein-protein interactions involving the RAG proteins alone. Neither protein shows biochemical activity alone, and the two proteins together are capable of cleaving the substrate DNA, not only at single signals, but in a concerted manner at pairs of signals. Protein-protein interactions are likely to be involved. (3) Study protein and DNA interactions in the putative larger complex. Mutant cells, defective in certain DNA repair proteins, are unable to complete the V(D)J recombination reaction. The proteins recognized by this behavior are potential participants in later steps in the recombination reaction. Since one candidate protein for this complex is a kinase, particular attention will be directed to phosphorylation events that may accompany complex formation. This recombination reaction is an essential step in the development of a normal immune system. Furthermore, errors in this reaction have been implicated in creating the chromosomal translocations that seem to precipitate many childhood leukemias. Finally, this metabolic pathway is connected to DNA break repair and cell cycle regulation which gives the study of V(D)J recombination a broader significance.
